Amino-substituted heterocycles as renin inhibitors

ABSTRACT

The invention concerns novel renin-inhibitory compounds which contain an amino-substituted heterocycle at the P 2  position. These are useful for treating renin-associated hypertension, congestive heart failure, glaucoma, hyperaldosteronism, and diseases caused by retroviruses including HTLV-I and -III. Processes for preparing the compounds, compositions containing them, and methods of using them are included. Also included is a diagnostic method which uses the compounds to determine the presence of renin-associated hypertension, or hyperaldosteronism.

This is a division of U.S. application Ser. No. 07/511,271 filed Apr.25, 1990, now U.S. Pat. No. 5,238,923, which is a Continuation-In-Partof U.S. application Ser. No. 07/357,561, filed May 26, 1989, nowabandoned.

BACKGROUND OF THE INVENTION

Renin is a natural enzyme which is released into the blood from thekidney. It cleaves its natural substrate, angiotensinogen, releasingdecapeptide, angiotensin I. This is in turn cleaved by converting enzymein the lung, kidney and other tissues to an octapeptide, angiotensin II.Angiotensin II raises blood pressure both directly by causing arteriolarconstriction and indirectly by stimulating release of thesodium-retaining hormone aldosterone from the adrenal gland causing arise in extracellular fluid volume. Inhibitors of renin have been soughtas agents for control of hypertension, congestive heart failure, andhyperaldosteronism.

European Application Number EP-229667 covers renin inhibitingpeptidyl-amino-diols of formula ##STR1## wherein A is a substituent; Wis C=O or CHOH; U is CH₂ or NR₂ ; R₁ is lower alkyl, cycloalkylmethyl,benzyl, 4-methoxybenzyl, halobenzyl, (1-naphthyl)methyl,(2-naphthyl)methyl, (4-imidazoyl)methyl, α,α-dimethylbenzyl,1-benzyloxyethyl, phenethyl, phenoxy, thiophenoxy or anilino; R₃ islower alkyl, [(alkoxy)-alkoxy]alkyl, (thioalkoxy)alkyl, lower alkenyl,benzyl or heterocyclic ring substituted methyl; R₄ is lower alkyl,cycloalkylmethyl or benzyl; R₅ is vinyl, formyl, hydroxymethyl orhydrogen; R₇ is hydrogen or lower alkyl; R₈ and R₉ are independentlyselected from OH and NH₂ ; and R₆ is hydrogen, lower alkyl, vinyl orarylalkyl.

European Application 186,977 covers certain renin-inhibitory peptides offormula ##STR2## wherein m is 0 or 1 and R¹ to R³ are a variety oforganic groups. The R³ covers many groups.

Structurally the positions of the various amino acids (amino acidmimics) of the compounds of the instant invention may be designated byreference to the octapeptide which is the minimal angiotensinogensequence cleaved by renin, namely: ##STR3##

A designation for the compounds of this invention is illustrated below.The CAD is considered to occupy the P₁ -P₁ ' positions. For example##STR4##

The present invention concerns novel compounds which inhibit renin. Italso concerns pharmaceutical compositions containing these novelpeptides, methods of treating renin-associated hypertension, congestiveheart failure, glaucoma, and hyperaldosteronism, as well as the use ofthe compounds as diagnostic tools, and the methods for preparing thecompounds.

Since HIV protease, like renin, is an aspartyl protease, these compoundscan also be used to treat diseases caused by retroviruses includingHTLV-I and -III.

SUMMARY OF THE INVENTION

The present invention relates to novel compounds of the formula ##STR5##and the pharmaceutically acceptable acid addition salts thereof whereinA, B, C, D, E, n, X, and Y are as defined herein below.

The invention also includes pharmaceutical compositions comprising aneffective amount of the above compound of formula I in admixture with apharmaceutically acceptable carrier or excipient and a method fortreating renin-associated hypertension in a patient suffering therefromcomprising administering to said patient the above pharmaceuticalcomposition in unit dosage form.

Further the invention includes a pharmaceutical composition comprisingan effective amount of a compound of formula I above in admixture with apharmaceutically acceptable carrier or excipient, and a method fortreating hyperaldosteronism in a patient suffering therefrom comprisingadministering to said patient the above pharmaceutical composition inunit dosage form.

Further the invention includes a pharmaceutical composition comprisingan effective amount of a compound of formula I in admixture with apharmaceutically acceptable carrier or excipient, and a method fortreating congestive heart failure in a patient suffering therefromcomprising administering to said patient the above pharmaceuticalcomposition in unit dosage form.

The present invention also includes the use of compounds of formula I totreat diseases caused by retroviruses.

The present invention also includes the use of compounds of formula Iabove as diagnostic tools for the identification of cases ofhypertension due to renin excess.

The present invention further includes a pharmaceutical compositioncomprising an amount effective for treating glaucoma. Of a compound ofFormula I in admixture with a pharmaceutically acceptable carrier orexcipient and a method for treating glaucoma in a patient sufferingtherefrom comprising administering to said patient the abovepharmaceutical composition in unit dosage form.

The invention further includes methods for preparing compounds offormula I above.

DETAILED DESCRIPTION

The following table provides a dictionary of the terms used in thedescription of the invention.

                  TABLE I                                                         ______________________________________                                        Abbreviated                                                                   Designation                                                                   ______________________________________                                                      Amino Acid                                                      GLU           L-Glutamic Acid                                                 ASP           L-Aspartic Acid                                                 PHE           L-Phenylalanine                                                 TYR (OMe)     O-Methyl-L-tyrosine                                             TYR           L-Tyrosine                                                      HIS           L-Histidine                                                     ATG           2-(2'-Amino-4'-thiazolyl)-                                                    glycine                                                         ATM           3-(2'-Amino-4'-thiazolyl)-                                                    alanine                                                         ATE           4-(2'-Amino-4'-imidazolyl)-                                                   ethylglycine                                                    AHM           3-(2'-Amino-4'-imidazoyl)-                                                    alanine                                                         AHY           4-(2'-Amino-4'-imidazoyl)-                                                    ethylglycine                                                                  C-Terminal Group                                                CAD                                                                                          ##STR6##                                                       CST                                                                                          ##STR7##                                                       STA                                                                                          ##STR8##                                                       CDH                                                                                          ##STR9##                                                       FCO                                                                                          ##STR10##                                                      FCS                                                                                          ##STR11##                                                                    Miscellaneous Groups                                            Z             Benzyloxycarbonyl                                               BOC           Tert-butyloxycarbonyl                                           AC            Acetyl                                                          CHO           Formyl                                                          TROC          Cl.sub.3 CCH.sub.2 OCO                                          BBSP          2-Benzyl-3-(t-butylsulfonyl)                                                  propionyl                                                       OBZL          Benzyl ester                                                    MPS                                                                                          ##STR12##                                                      SMO                                                                                          ##STR13##                                                      DMSA                                                                                         ##STR14##                                                      SPI                                                                                          ##STR15##                                                      AEM                                                                                          ##STR16##                                                      BHEAEA                                                                                       ##STR17##                                                      MBA                                                                                          ##STR18##                                                      PIP                                                                                          ##STR19##                                                                    Solvents and Reagents                                           Et.sub.2 O    Diethyl ether                                                   CHCl.sub.3    Chloroform                                                      DMF           N,N-Dimethylformamide                                           DMSO          Dimethylsulfoxide                                               HOBT          Hydroxybenzotriazole                                            DCC           N,N'-Dicyclohexylcarbodiimide                                   HOAc          Acetic acid                                                     Et.sub.3 N    Triethylamine                                                   THF           Tetrahydrofuran                                                 CH.sub.2 Cl.sub.2                                                                           Dichloromethane                                                 MeOH          Methanol                                                        EtOAc         Ethyl acetate                                                   DMAP          4-(N,N-Dimethylamino)pyridine                                   ______________________________________                                    

The compounds of the present invention are represented by the formula##STR20## or a pharmaceutically acceptable acid addition salt thereof,wherein

A is H, BOC, BBSP, Z, ##STR21##

wherein R and R₁ are each independently hydrogen or straight or branchedchain lower alkyl which is unsubstituted or substituted by one or twohydroxy groups, one or two amino groups or ##STR22## wherein this is asaturated ring containing two to five carbon atoms wherein

Q is CH₂, O, S, or NR;

B is absent, PHE, TYR, or TYR(OMe) with the proviso that when A is BBSP,B is absent;

C is CST, FCS, FCO, CAD or STA;

D is absent, OH, NR₂ R₃ wherein R₂ and R₃ are each independentlyhydrogen or straight or branched lower alkyl or when R₂ is hydrogen R₃can also be --(CH₂)_(m) X wherein m is an integer of from zero to eightand X is --OH, ##STR23## as defined above, OR₄, NR₅ R₆ wherein R₄, R₅,and R₆ are each independently hydrogen, straight or branched chain loweralkyl, substituted or unsubstituted by one or two hydroxy or aminogroups with the proviso that when C is CAD, D is absent;

E is hydrogen, Z, BOC, or lower alkanoyl,

n is an integer of from 0 to 2;

X and Y are each independently O, S, N or NH and at least one of X and Ymust be N; X and Y cannot both be N.

Preferred compounds of the present invention are those of formula Iwherein

A is BOC, BBSP, ##STR24##

B is absent, PHE, or TYR(OMe),

C is CST, FCO, FCS or CAD;

D is absent or ##STR25##

with the proviso that when C is CAD D is absent;

E is hydrogen or lower alkanoyl; and n is one.

More preferred compounds of the present invention are those of formula Iwherein

A is ##STR26##

B is PHE or TYR(OMe).

Still more preferred compounds of the present invention are those offormula I wherein

A is ##STR27##

B is PHE,

C is CST or CAD, and

E is hydrogen.

Particularly preferred compounds falling within the scope of theinvention include the following compounds, their isomers, andpharmaceutically acceptable acid addition salts:

SPI-PHE-ATM(Z)-CAD,

MPS-PHE-ATM(Z)-CAD,

DMSA-PHE-ATM(Z)-CAD,

BBSP-ATM(Z)-CAD,

BBSP-ATM (AC)-CAD,

BBSP-ATM-CST-AEM,

BBSP-ATM-CST-BHEAEA,

SP X -TYR (OME)-ATM (Z)-CAD,

SP X-TYR (OME)-ATM (CHO)-CAD,

BBSP-ATM-FCO-AEM,

DMSA-TYR(OME)-ATM-FCS-AEM,

BOC-PHE-ATM-CAD,

BOC-PHE-ATM-CST-AEM,

BOC-PHE-ATM-CST-BHEAEA,

BBSP-ATG-CAD,

DMSA-PHE-ATG-CST-AEM,

SPI-TYR (OME)-ATG-CAD,

SPI-PHE-ATG-CAD,

SMO-TYR (OME)-ATG-CST-AEM,

SMO-PHE-ATE-CAD,

SMO-TYR(OME)-ATE-CAD,

SMO-PHE-AHM-CAD,

SMO-PHE-AHM-CST-BHEAEA,

DMSA-TYR(OME)-AHY-CST-AEM,

SMO-PHE-ATM-FCS-AEM,

SMO-PHE-ATM-FCO-AEM,

SPI-PHE-ATM-CAD,

BOC-ATG(Z)-FCS-AEM,

ATG(Z)-FCS-AEM,

SMO-PHE-ATG(Z)-FCS-AEM,

SMO-PHE-ATG-FCS-AEM,

SMO-PHE-ATM(TROC)-FCS-OET,

SMO-PHE-ATM (TROC)-FCO-OET,

SMO-PHE-ATM-FCO-OET, and

SMO-PHE-ATG(Z)-FCS-AEM.

Most preferred compounds are:

SMO-PHE-ATM(Z)-CAD,

SMO-PHE-(S)ATM-CAD (Isomer A),

SMO-PHE-ATM-CST-AEM,

SMO-TYR(OME)-ATM-CST-AEM,

SMO-PHE-ATM-CAD (Isomer B),

SMO-TYR(OME)-ATM-CAD,

SMO-PHE-ATG-CAD,

BOC-PHE-ATG-STA-MBA, and

SMO-PHE-ATM-CST-BHEAEA. The P₂ in the present invention may have asubstituent (E): E=(Z), (AC) or (CHO), represented by the followingabbreviation; ATM(E). The substituent is on the exocyclic nitrogen asshown by ##STR28##

The compounds include solvates and hydrates and pharmaceuticallyacceptable acid addition salts of the basic compounds of formula Iabove.

The term pharmaceutically acceptable acid addition salt is intended tomean a relatively non-toxic acid addition salt either from inorganic ororganic acids such as, for example, hydrochloric, hydrobromic,hydroiodic, sulfuric, phosphoric, acetic, citric, oxalic, malonic,salicylic, malic, benzoic, gluconic, fumaric, succinic, ascorbic,maleic, tartaric, methanesulfonic, and the like. The salts are preparedby contacting the free base form with a sufficient amount of the desiredacid to produce a salt in the conventional manner. The free base formsmay be regenerated by treating the salt form with a base.

The compounds of the present invention possess one or more chiralcenters and each center may exist in the R(D) or S(L) configuration. Thepresent invention includes all enantiomeric, epimeric and tautomericforms as well as the appropriate mixtures thereof.

The S isomer at the P₂ position is the more preferred.

Some of the above novel compounds may be prepared in accordance withwell-known procedures for preparing compounds from their constituentamino acids. Other of the novel compounds of the present invention areprepared by a step-wise procedure or by a fragment coupling proceduredepending upon the particular final product desired.

One process for preparing a compound of formula I comprises:

a) reacting an N-protecting amino acid with a desired amine to producethe corresponding amide,

b) deprotecting the N-protecting group of said amide and coupling itwith a desired acid to produce a dipeptidyl like amide, and

c) further deprotecting the side chain functions on the amide to producethe desired compound of formula I and converting, if desired, to apharmaceutically salt thereof.

An alternate process for preparing a compound of formula I wherein C isFCS or FCO comprises:

a) reacting an amino acid ester with TROC protection on the side chainwith an N-protected amino acid to produce a dipeptide ester which isthen hydrolyzed to the corresponding dipeptide acid,

b) coupling the product of step a) with an amine selected from the groupconsisting of FCS or FCO to produce the TROC-protected compound of claim1, and

c) deprotecting further, if desired, to produce a compound of claim 1and converting, if desired, to a pharmaceutically acceptable saltthereof.

This is illustrated in Scheme II below.

The following schemes illustrate novel methods of preparing certaincompounds of the present invention.

According to Scheme I below, α-BOC, (2'-(Z)-amino-4'-thiazolyl)alanine(1) is reacted with CST-AEM (2) to form an amide (3). The reaction takesplace in an inert solvent such as CH₂ Cl₂ or DMF with HOBT and DCC attemperatures of -20° to 30° C. The BOC protecting group of (3) isremoved with HCl gas or TFA in an inert solvent such as CH₂ Cl₂ to givethe amine (4). This amine (4) is coupled with SMO-PHE (5) in an inertsolvent such as CH₂ Cl₂ or DMF with HOBT and DCC at temperatures from-20° to 30° C. to form (6), a compound of the present invention.Furthermore, hydrogenolysis of (6) (removal of Z) by stirring inmethanol under a hydrogen atmosphere in the presence of 10-20% palladiumon carbon catalyst, with or without the presence of p-toluenesulfonicacid, forms (7) a compound of the present invention.

Scheme I can be modified to include the (TROC) protecting group in placeof (Z) in order to improve the synthetic route and provide for thepreparation of compounds containing FCO and FCS. The TROC protectinggroup is useful as it is easier to remove in going from step (6) to (7).The FCO or FCS group is more stable in this situation. ##STR29##

The strategy of peptide chain assembly and selection and removal ofprotecting groups is discussed in Chapter 1, "The Peptide Bond," in "ThePeptides. Analysis, Synthesis, Biology," E. Gross and J. Meienhofer,Eds., Academic Press, New York, N.Y., 1979, Vol. 1, pp. 42-44.

The DCC/HOBT method of coupling is well-known to those skilled in theart and is discussed in Chapter 5, "The Carbodiimide Method" by D. H.Rich and J. Singh in "The Peptides. Analysis, Synthesis, Biology," E.Gross and J. Meienhofer, Eds., Academic Press, New York, N.Y., 1979,Vol. 1, pp. 241-261.

Peptide coupling depends on activating the carboxy terminus of the aminoprotected amino acid and condensing it with another peptide containing afree amino terminus. In addition to the DCC coupling method describedabove, other methods of activating the carboxyl group of a protectedamino acid include: 1) The azide method--described in Chapter 4 of theabove reference. 2) The mixed anhydride method--described in Chapter 6of the above reference. 3) The active ester method--described in Chapter3 of the above reference.

The term lower alkyl refers to straight or branched chain alkyl radicalscontaining from one to six carbon atoms including but not limited tomethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl,2-methylhexyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl,2-methylpentyl, 2,2-dimethylpropyl, n-hexyl, and the like.

The term lower alkanoyl means refers to alkanoyl groups of from one tofour carbon atoms.

The compounds of the present invention are useful for treatingrenin-associated hypertension, congestive heart failure,hyperaldosteronism, and other related illnesses. They are useful asagents in treating glaucoma. They are also useful as diagnostic toolsfor determining the presence of renin-associated hypertension orhyperaldosteronism.

Pharmaceutical compositions which comprise an effective amount of thecompound in combination with a pharmaceutically acceptable carrier arepart of the present invention. An important aspect of the presentinvention is a method of treating renin-associated hypertension in amammal which comprises administering a pharmaceutical compositioncontaining an effective amount of a compound of the invention incombination with a pharmaceutically acceptable carrier to the mammal.

Another equally important aspect of the present invention is a method oftreating hyperaldosteronism in a mammal which comprises administering apharmaceutical composition containing an effective amount of a compoundof the invention in combination with a pharmaceutically acceptablecarrier to the mammal.

An additional aspect of the present invention is a method for treatingcongestive heart failure in a mammal which comprises administering apharmaceutical composition containing an effective amount of a compoundin combination with a pharmaceutically acceptable carrier to the mammal.

Yet another aspect of the present invention is a process for preparing acompound of formula I according to claim 1.

The effectiveness of the aforementioned compounds is determined by atest for in vitro renin inhibitory activity. This activity is determinedby a standard radioimmunoassay for angiotensin I. In this assay theenzyme, renin, incubated for 2 hours at 37° C. in the presence of asubstrate, angiotensinogen, generates the product, angiotensin I. Testcompounds are added to the incubation mixture. Relative activity isreported as the IC₅₀ , which is the molar concentration of test compoundcausing a 50% inhibition of the renin activity.

The compounds of the present invention have the advantages of increasedstability toward chymotrypsin hydrolysis, which is described by J MedChem, Vol. 31, No. 2, page 292, 1988. This property makes the compoundsmore stable in vivo and therefore they exhibit a longer duration of invivo activity.

Compounds of this invention have also demonstrated in vivo activityrepresented by lowering blood pressure in conscious monkeys. In vivoeffectiveness is determined by their effect on blood pressure inunanesthetized, sodium-deplete, normotensive Rhesus or Cynomolgusmonkeys.

The following describes this test. Monkeys were acclimated to a lowsodium diet and trained to rest quietly in a restraining device. Next,vascular access ports were surgically implanted for intravenousadministration of test compounds and direct measurement of bloodpressure. At least one week was allowed for recovery from surgery beforesodium depletion was accomplished by giving furosemide (1 mg/kg/day, IM)for 4 consecutive days. On the seventh day animals were removed fromtheir home cage and placed in the restraining device. After a 20- to30-minute acclimation period, a control blood sample (arterial) wastaken for determination of plasma renin activity (PRA). Next, eithervehicle (absolute ethanol, 0.2 mL/kg) or test compound (5 mg/kg) wasinfused intravenously over a 10-minute period.

Blood pressure was monitored continuously throughout the entire pre- andpost-dose period. Blood samples were taken at the mid-point of theinfusion and at 0, 15, 30, and 60 minutes post infusion.

The compounds of the present invention also possess the advantage ofincreased selectivity toward the renin enzyme versus other aspartylprotease enzymes.

                  TABLE II                                                        ______________________________________                                        In Vitro Renin Inhibition                                                                               IC.sub.50 (nM,                                                                or % inhi-                                                                    bition at con-                                      Compound                  centration)                                         ______________________________________                                        SMO--PHE--ATM--CAD (Isomer A)                                                                           0.38                                                SMO--PHE--ATM--CAD (Isomer B)                                                                           0.76                                                (impure sample)                                                               SMO--PHE--ATM(Z)--CAD     0% at 10.sup.-8                                     BOC--PHE--ATM--CST--AEM   15% at 10.sup.-8                                    SMO--PHE--ATM--STA--MBA   480                                                 BOC--PHE--ATG--STA--MBA   44% at 10.sup.-6                                    SMO--PHE--ATM--CST--AEM (Isomer A)                                                                      49.9% at 10.sup.-6                                  SMO--PHE--ATM--CST--AEM (Isomer B)                                                                      3.4                                                 SMO--TYR(OME)--ATM--CAD (Isomer A)                                                                      0.34                                                SMO--TYR(OME)--ATM--CAD (Isomer B)                                                                      66                                                  SMO--PHE--ATM(Z)--CST--AEM (Isomer A)                                                                   11.4% at 10.sup.-8                                  SMO--PHE--ATM(Z)--CST--AEM (Isomer B)                                                                   25.5% at 10.sup.-8                                  SMO--PHE--ATG--CAD        0.58                                                SMO--PHE--ATM(Z)--FCS--AEM                                                                              164                                                 SMO--PHE--ATM--FCS--AEM   0.58                                                SMO--PHE--ATM--CDH        0.27                                                SMO--PHE--ATE--CAD        15.3                                                SMO--PHE--ATE(Z)--CAD     NA at 10.sup.-8                                     SMO--PHE--ATG(Z)--FCS--AEM                                                                              NA at 10.sup.-8                                     SMO--PHE--ATM--FCO--AEM                                                       SMO--PHE--ATM--CST--BHEAEA                                                                              3.6                                                 SPI--PHE--ATM--CAD        0.16                                                ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        In Vitro Chymotrypsin Stability                                                                 Percent Parent                                                                Remaining (3 hrs)                                                             Buffer Buffer +                                                               only   chymotrypsin                                         ______________________________________                                        SMO--PHE--(S)ATM--CAD                                                                             100      100                                              SMO--TYR(OME)--ATM--CAD                                                                           100      100                                              (Isomer A)                                                                    ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        In Vivo Blood Pressure Lowering by Renin Inhibitors                           SMO--PHE--(S)ATM--CAD                                                                   (mm Hg drop in mean B.P.)                                           PO Dose  n at   1 hr     2 hr 4 hr   6 hr Max                                 ______________________________________                                        10 mg/kg 4      14       20   15     12   23                                  30 mg/kg 6      30       34   29     29   38                                  ______________________________________                                    

As can be seen from the above tables, the compounds of the presentinvention have a significant effect on the activity of renin and thusare useful for the treatment of hypertension, hyperaldosteronism, andcongestive heart failure.

The compounds of the instant invention, when tested by measuring theeffect on intraocular pressure in rabbits as described by Tinjum, A.M.,Acta Ophthalmologica 50, 677 (1972), are expected to inhibitantiglaucoma activity.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets, and suppositories. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be encapsulatingmaterial. In powders, the carrier is a finely divided solid which is inadmixture with the finely divided compound. In the tablet the activecompound is mixed with carrier having the necessary binding propertiesin suitable proportions and compacted in the shape and size desired. Thepowder and tablets preferably contain from 5 to 10 to about 70 percentof the active ingredient. Suitable solid carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, tragacanth, methylcellulose,a low melting wax, cocoa butter, and the like. The term "preparation" isintended to include the formulation of the active compound withencapsulating material as carrier providing a capsule in which theactive component (with or without other carriers) is surrounded bycarrier, which is thus in association with it. Similarly, cachets areincluded. Tablets, powders, cachets, and capsules can be used as soliddosage forms suitable for oral administration.

The compound of the present invention may be administered orally,buccally, parenterally, by inhalation spray, rectally, or topically indosage unit formulations containing conventional nontoxicpharmaceutically acceptable carriers, adjuvants and vehicles as desired.The term parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques.

For preparing suppositories, a low melting wax such as a mixture offatty acid glycerides or cocoa butter is first melted, and the activeingredient is dispersed homogeneously therein by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby solidify.

Liquid form preparations include solutions, suspensions, and emulsions.As an example may be mentioned water or water/propylene glycol solutionsfor parenteral injection. Liquid preparations can also be formulated insolution in aqueous polyethyleneglycol solution. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, i.e., natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions, and emulsions. These particular solid form preparations aremost conveniently provided in unit dosage form and as such are used toprovide a single liquid dosage unit. Alternately, sufficient solid maybe provided so that after conversion to liquid form, multiple individualliquid doses may be obtained by measuring predetermined volumes of theliquid form preparation as with a syringe, teaspoon, or other volumetriccontainer. When multiple liquid doses are so prepared, it is preferredto maintain the unused portion of said liquid doses at low temperature(i.e., under refrigeration) in order to retard possible decomposition.The solid form preparations intended to be converted to liquid form maycontain, in addition to the active material, flavorants, colorants,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The liquid utilized forpreparing the liquid form preparation may be water, isotonic water,ethanol, glycerine, propylene glycol, and the like, as well as mixturesthereof. Naturally, the liquid utilized will be chosen with regard tothe route of administration, for example, liquid preparations containinglarge amounts of ethanol are not suitable for parenteral use.

Preferably, the pharmaceutical preparation is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, for example, packeted tablets, capsules, and powders invials or ampules. The unit dosage form can also be a capsule, cachet, ortablet itself, or it can be the appropriate number of any of these inpackaged form.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from 1 mg to 500 rag, preferably 5 to 100 mgaccording to the particular application and the potency of the activeingredient. The compositions can, if desired, also contain othercompatible therapeutic agents.

In therapeutic use as renin inhibitors, the mammalian dosage range for a70-kg subject is from 1 to 2500 mg per day or preferably 25 to 750 mgper day optionally in divided portions. The dosages, however, per daymay be varied depending upon the requirements of the patient, theseverity of the condition being treated, and the compound beingemployed. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmall dosages which are less than the optimum dose of the compound.Thereafter the dosage is increased by small increments until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

In therapeutic use as an antiglaucoma agent, the compound may also beadministered as a topical corneal application of a solution containingthe compound in amounts as known to one skilled in the treatment.

The present invention includes combinations of novel renin-inhibitingcompounds of formula I with one or more antihypertensive agents selectedfrom the group consisting of diuretics, α- and/or β-adrenergic blockingagents, calcium channel blocking agents, central nervous system-actingagents, adrenergic neuron blocking agents, vasodilators, angiotensinconverting enzyme inhibitors, and other antihypertensive agents.

The following examples are provided to enable one skilled in the art topractice the present invention. These examples are not intended in anyway to limit the scope of the invention but are illustrative thereof.

EXAMPLE 1 SMO-PHE-ATM(Z)-CAD

A mixture of SMO-PHE (0.786 g), HOBT (0.339 g), and DCC (0.516 g) wasstirred at 0° C. and treated with ATM(Z)-CAD (1.4 g). The mixture waswarmed to room temperature and stirred overnight. The reaction mixturewas filtered free of precipitate and the filtrate evaporated in vacuo.The residue was dissolved in ethyl acetate (100 mL) and washedsuccessively with 1M citric acid (100 mL), saturated NaHCO₃ (100 mL) andsaturated salt solution (100 mL). The organics were dried over MgSO₄ andevaporated in vacuo to give a yellow foam. The foam was chromatographedover silica gel to give 1.5 g of the product as a mixture of twodiastereomers; mp 95°-97° C.

Analyzed for C₄₁ H₅₈ N₆ O₉ S₂ ·0.5 CHCl₃

Calc'd: C, 55.82; H, 6.61; N, 9.43

Found: C, 56.06; H, 6,68; N, 9.15

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 2 SMO-TYR(OME)-ATM(Z)-CAD

The product was synthesized as in Example 1, from SMO-TYR(OME) (0.860g), HOBT (0.338 g), DCC (0.516 g), and ATM(Z)-CAD (1.4 g). This gave 1.3g of the product as a mixture of diastereomers, mp 101°-107° C.

Analyzed for C₄₂ H₅₆ N₆ O₉ S₂ ·0.5 ether

Calc'd: C, 58.06; H, 7.21; N, 9.23

Found: C, 58.39; H, 6.93, N, 9.05

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 3 BOC-PHE-ATM(Z)-CST-AEM

The product was synthesized as in Example 1 from BOC-PHE (0.451 g), HOBT(0.230 g), DCC (0,351 g), and ATM(Z)-CST-AEM (1.2 g ). This gave 1.2 gof product. This material was used to prepare the product of Example 4.The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 4 BOC-PHE-ATM-CST-AEM

BOC-PHE-ATM(Z)-CST-AEM (1.05 g) was slurried with 20% Pd/C (0.3 g) andp-toluenesulfonic acid (0.476 g) in methanol (25 mL) under a hydrogenatmosphere. The mixture was stirred for 2 hours, filtered free ofcatalyst, and evaporated in vacuo. The residue was dissolved in ethylacetate (75 mL). The organic phase was washed with sodium carbonate (75mL) and sodium chloride (50 mL), then dried over MgSO₄ and evaporated invacuo. The residue was co-evaporated once with methanol (10 mL) to give0.63 g of the product as a white foam as a mixture of diastereomers, mp95°-103° C.

Analyzed for C37H₅₇ N₇ O₇ S·0.9 MeOH

Calc'd: C, 58.90; H, 7.90; N, 12.69

Found: C, 59.15; H, 8.05; N, 12.37

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLES 5 AND 6 SMO-PHE-ATM(Z)-CST-AEM Isomer A (5) and Isomer B (6)

The product was synthesized as in Example 1 from SMO-PHE (0,817 g), HOBT(0,351 g), DCC (0,536 g), and ATM(Z)-CST-AEM (1.7 g) . This gave 2.0 gof product as a mixture of diastereomers which were separated bychromatography over silica gel. The fast eluting isomer is Isomer A.Isomer A (5) (0.64 g), mp 102°-110° C.

Analyzed for C₄₄ H₆₂ N₈ O₁₀ S₂ ·0.4 CHCl₃

Calc'd: C, 54.70; H, 6.45; N, 11.49

Found: C, 54.66; H, 6.64; N, 11.37

Isomer B (6) (0.63 g), mp 105°-110° C.

Analyzed for C₄₄ H₆₂ N₈ O₁₀ S₂ ·0.4 CHCl₃)

Calc'd: C, 54.70; H, 6.45; N, 11.49

Found: C, 54.46; H, 6.58; N, 11.56

Both isomers show consistent NMR and mass spectra.

EXAMPLE 7 SMO-PHE-ATM-CST-AEM (Isomer A)

The product was synthesized as in Example 4 from SMO-PHE-ATM(Z)-CST-AEM(Isomer A) (0.717 g), p-toluenesulfonic acid (0,366 g) and 20% Pd/C (0.3g).

This gave 0.427 g of product as a white solid, mp 109°-119° C.

Analyzed for C₂ H₅₆ N₈ O₈ S₂

Calc'd: C, 53.80; H, 7.35; N, 13.53

Found: C, 53.70; H, 7.33; N, 13.48

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 8 SMO-PHE-ATM-CST-AEM (Isomer B)

The product was synthesized as in Example 4 from SMO-PHE-ATM (Z)-CST-AEM(Isomer B) (0. 883 g ), p-toluenesulfonic acid (0.453 g) and 20% Pd/C(0.4 g). This gave 0.544 g of product as a white foam, mp 100°-113° C.

Analyzed for C36H₅₆ N₈ O₈ S₂ ·1.3 MeOH

Calc'd: C, 53.67; H, 7.39; N, 13.43

Found: C, 54.01; H, 7.40; N, 13.19

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLES 9 AND 10 SMO-PHE-ATM-CAD (Method A)

Isomer A, fast eluting (9);

Isomer B, slow eluting (10)

The product was synthesized as in Example 4 from SMO-PHE-ATM(Z)-CAD (4.8g), p-toluenesulfonic acid (2.182 g), and 20% Pd/C (0.4 g). This gave0.91 g of a mixture of diastereomers as a white foam, which wereseparated by chromatography over silica gel.

Isomer A (1.50 g), mp 104°-112° C.

Analyzed for C₃₃ H₅₂ N₆ O₇ S₂ ·0.1CH₂ Cl₂ ·0.4 EtOAc

Calc'd: C, 55.37; H, 7.42; N, 11.17

Found: C, 55.40; H, 7.28; N, 10.94

Isomer B (1.30 g), mp 109°-122° C.

Analyzed for C₃₃ H₅₂ N₆ O₇ S₂ ·0.1CH₂ Cl₂ ·0.4 EtOAc

Calc'd: C, 55.37; H, 7.42; N, 11.17

Found: C, 55.09; H, 7.25; N, 10.86

Both isomers showed consistent NMR and mass spectra.

EXAMPLE 9 (Method B) SMO-PHE-ATM-CAD, Isomer A or SMO-PHE-(S)ATM-CAD

The product was synthesized as in Example 1 from SMO-PHE (0.628 g),Et,N: (0.505 g), HOB7 (0.270 g), DCC (0.413 g) and (S)-ATM-CAD·2HCl (1.1g). The crude product was chromatographed over silica gel to give 0.375g of the pure compound as a single diastereomer, mp 106°-113° C.

Analyzed for C₃₃ H₅₂ N₆ O₇ S₂ ·0.3 EtOAc

Calc'd: C, 55.86; H, 7.46; N, 11.43

Found: C, 55.51; H, 7.50; N, 11.37

The compound showed consistent NMR and mass spectra.

EXAMPLE 9 (Method C) SMO-PHE-(S)ATM-CAD

A solution of 10.60 g SMO-PHE-(S)ATM(TROC)-CAD (12.0 mmol) was dissolvedin 250 mL 4:1, THF (freshly distilled)/MeOH, in a 500 mL roundbottomflask at room temperature under a nitrogen atmosphere. To this was added6.41 g ammonium chloride 10 equivalents, and 1.57 g zinc dust, 2equivalents. The reaction was stirred vigorously at ambient temperature.After 2 hours another two equivalents of zinc was added. After another 2hours, another 2 equivalents for a total of 6 equivalents zinc. After5.5 hours the reaction mixture was filtered and concentrated to afford10.96 g white foam. The reaction mixture was chromatographed (SiO₂, 1:1ethyl acetate/CH₂). A solid of 1.02 g was filtered from the sample priorto introduction onto the column. Product of 6.47 g was isolated. Massspectrum m/e=709.2.

EXAMPLES 11 AND 12 SMO-TYR (OME)-ATM-CAD Isomer A, fast eluting (11);Isomer B, slow eluting (12)

The product was synthesized as in Example 4 from SMO-TYR(OME)-ATM(Z)-CAD(1.4 g), p-toluenesulfonic acid (0.610 g) and 20% Pd/C (0.25 g). Thisgave 1.1 g of a mixture of diastereomers as a white foam. The isomerswere separated by chromatography over silica gel.

Isomer A (0.330 g), mp 110°-120° C.

Analyzed for C₃₄ H₅₄ N₆ O₈ S₂ ·MeOH·H₂ O

Calc'd: C, 53.28; H, 7.66; N, 10.65

Found: C, 53.39; H, 7.23; N, 10.41

Isomer B (0.240 g), mp 106°-116° C.

Analyzed for C₃₄ H₅₄ N₆ O₈ S₇ ·1.5 MeOH

Calc'd: C, 54.17; H, 7.68; N, 10.68

Found: C, 54.24; H, 7.36; N, 10.67

Both isomers showed consistent NMR and mass spectra.

EXAMPLE 13 SMO-PHE-ATM(Z)-STA-MBA

The product was formed as in Example 1 from SMO-PHE(0.517 g), HOBT(0,222 g), DCC (0.339 g), and ATM(Z)-STA-MBA (0.90 g). This gave 1.1 gof product as a mixture of diastereomers as a white foam, mp 97°-103° C.

Analyzed for C₄₀ H₅₇ N₇ O₉ S₂ ·1.5 MeOH

Calc'd: C, 55.87; H, 7.11; N, 10.99

Found: C, 55.51; H, 6.71; N, 10.83

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 14 SMO-PHE-ATM-STA-MBA

The product was synthesized as in Example 4 from SMO-PHE-ATM(Z)-STA-MBA(1.0 g), p-toluenesulfonic acid (0.228 g) and 20% Pd/C (0.3 g). Thisgave 0.399 g of product as a mixture of diastereomers, mp 97°-108° C.

Analyzed for C₃₂ H₅₁ N₇ O₇ S₂ ·0.65 Et₂

Calc'd: C, 54.82; H, 7.65; N, 12.94

Found: C, 54.73; H, 7.57; N, 12.97

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 15 BOC-PHE-ATG(Z)-STA-MBA

The product was synthesized as in Example 1 from ATG(Z)-STA-MBA (1.6 g),HOBT (0.419 g), DCC (0.640 g), and BOC-PHE (0.796 g). This gave 1.6 g ofproduct as a mixture of diastereomers as a light yellow foam, mp108°-118° C.

Analyzed for C₄₀ H₅₆ N₆ O₈ S (99%)

Calc'd: C, 60.89; H, 7.16; N, 10.65

Found: C, 60.98; H, 7.19; N, 10.55

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 16 BOC-PHE-ATG-STA-MBA

The product was synthesized as in Example 4 from BOC-PHE-ATG(Z)-STA-MBA(1.5 g), p-toluenesulfonic acid (0.38 g) and 10% Pd/C. This gave 0.31 gof product as a mixture of diastereomers as a white solid, mp 174°-176°C.

Analyzed for C₃₂ H₅₀ N₆ O₆ S (99%)

Calc'd: C, 59.79; H, 7.72; N, 12.87

Found: C, 59.97; H, 7.99; N, 13.06

The structure was confirmed by NMR and mass spectroscopy.

EXAMPLE 17 TROC-SPI-PHE-(S)ATM(TROC)-CAD

A solution of 2.10 g (TROC) SPI-PHE (4.30 mmol) in 30 mL DMF was treatedwith 0.89 g DCC (4.30 mmol) and 0.58 g HOBT (4.30 mmol). The materialswere allowed to stir at 15° C. for 30 minutes. Then 2.50 g of(S)ATM(TROC)-CAD (4.25 mmol) in 10 mL MDF was added and the ice bathremoved. The mixture continued stirring at RT for 48 hours. The reactionmixture was filtered and evaporated in vacuo. The residue was taken upin CH₂ Cl₂ and washed with 5% Na₂ CO₃ and saturated NaCl. The organiclayer was dried over MgSO₄ and evaporated. Chromatography on silica geleluting with a gradient of straight CHCl₃ to 2% MeOH in CHCl₃ gave 3.95g of product. Structure was confirmed by NMR spectroscopy.

EXAMPLE 18 SPI-PHE-(S)ATM-CAD

A solution of 2.00 g TROC-SPI-PHE-(S)ATM-(TROC)-CAD (1.89 mmol) in a 1:1mixture of 40 mL HOAc:MeOH was treated with 3.50 g zinc dust. Thesematerials were sonicated at ambient bath temperature under N₂ atmospherefor 7 hours. The reaction mixture was diluted with Et₂ O, filtered, andevaporated. The residue was taken up in EtOAc and washed twice withsaturated NaHCO₃. The organic layer was dried over MgSO₄, andevaporated. Chromatography on silica gel eluting with a gradient 2 to 8%MeOH in CHCl₃ gave 0.47 g of the product. The structure was confirmed byNMR and mass spectroscopy.

Analyzed for for C₃₃ H₅₃ N₇ S₂ O₆ ·1.0 CH₂ Cl₂ (MW 792.89):

Calc'd: C, 51.54; H, 6.99; N, 12.37

Found: C, 51.21; H, 6.77; N, 12.26.

EXAMPLE 19 SMO-PHE-(S)ATM(TROC)-CDH

A solution of 0.89 g (BOC)-(S)ATM(TROC)-CDH was dissolved in CH₂ Cl₂ andcooled in an ice bath. HCl was bubbled through for 10 minutes, and thereaction stirred an additional 30 minutes, then concentrated to give0.91 g off-white foam. This was redissolved in CH₂ Cl₂ and cooled, and0.42 g SMO-PHE, 0.27 g DCC, 0.18 g HOBT, and 0.48 g DMAP added. Aftercoming to ambient temperature overnight, the reaction mixture wasfiltered and washed sequentially with 5% citric acid, saturated NaHCO₃,and saturated NaCl, then concentrated. The 1.12 g solid waschromatographed (SiO₂, EtOAc), and 0.71 g product collected. Massspectrum m/e=872.

EXAMPLE 20 SMO-PHE-(S)ATM-CDH

A compound of 0.68 SMO-PHE-(S)ATM(TROC)-CDH was dissolved in 4:1THF/MeOH. Ammonium chloride of 0.35 g and 0.10 g zinc dust were addedand the reaction stirred at room temperature. After 1 hour another 0.10g zinc was added, and another 0.10 g zinc after 2 hours. After 5 hoursthe reaction mixture was filtered and concentrated. The residue wasdissolved in methanol and 10 mL HOAc added, along with 0.30 g zinc. Thereaction was heated; after 45 minutes another 0.30 g zinc and reactionstirred for an hour. After cooling the mixture was filtered, dilutedwith Et₂, and poured in NaHCO₃ solution. The layers were separated, theaqueous washed with more ether, and the combined organics concentratedto give ca. 1.3 g glass. This acid sequence was repeated, and worked upby concentration of the filtrate, dissolving in EtOAc and a NaHCO³ wash.Concentration of the organics gave 0.41 g solid which waschromatographed (SiO₂, 95% CHCl₃ 5% MeOH). Product of 0.32 g wasobtained. Mass spectrum m/e=695.

EXAMPLE 21 SMO-PHE-(S)ATM(TROC)-CAD

A solution of 32.93 g (BOC)-(S)ATM(TROC)-CAD (47.8 mmol), milled, wassuspended in ether/methanol (300 mL/20 mL) and cooled in ice water bath.HCl was bubbled through for 20 minutes. An initial white precipitatewent back into solution during this time. The reaction mixture wasstored in the refrigerator overnight. The reaction mixture wasconcentrated, the foam stirred vigorously with ether and the resultingwhite solid filtered and dried to afford 27.5 g powder (44.0 mmol, 92%as amine hydrochloride, subsequent calculations based on this). Thematerial was used without further purification. This amine was suspendedin 500 mL CH₂ CL₂, the flask cooled in ice water bath, and 13.83 gSMO-PHE, 5.96 g HOBT, 5.83 g DMAP, and 9.06 g DCC added. The mixture wasallowed to come to ambient temperature overnight. It was filtered, andthe filtrate washed sequentially with water, 5% aqueous citric acid,saturated NaHCO₃, and saturated NaCl, then dried over Na₂ SO₄ andconcentrated to give 31.23 g white foam. This material waschromatographed in two lots (SiO₂, eluting with 2:1 ethylacetate/hexane). Total 11.38 g desired product isolated. Mass spectrumm/e=885.

EXAMPLE 22 SMO-PHE-(S) ATM(TROC)-FCS-AEM

To SMO-PHE (0.87 g, 2.46 mmol) and HOBt (0.37 g, 2.76 mmol) in anhydrousDMF (20 mL) at O° C. was added DCC (0.57 g, 2.76 mmol ) in DMF (5 mL )followed by (S)ATM(TROC)-FCS-AEM (1.95 g, 2.76 mmol) in DMF (5 mL).After stirring for 2 hours, the reaction was allowed to warm to roomtemperature and stirred for a further 16 hours. The precipitateddicyclohexylurea was removed by filtration and the solvent evaporatedunder reduced pressure. The crude residue was taken up in ethyl acetateand washed with saturated aqueous sodium bicarbonate followed by brine.After drying (Na₂ SO₄), filtration and evaporation of the solvent thecrude product was chromatographed on silica gel eluting with 5% methanolin dichloromethane. The structure of the major product (1.35 g, 49%),obtained as a white foam, was confirmed by NMR and mass spectroscopy; MS(FAB); MH⁺ 1005.3; (HPLC-98% purity).

Analyzed for C₃₉ H₅₅ N₈ O₁₀ S₂ O₃ F₂ ·0.2 CH₂ Cl₂

Calc'd: C, 46.09; H, 5.42; N, 10.97; S, 6.28

Found: C, 45.77; H, 5.55; N, 10.82; S, 6.09

EXAMPLE 23 SMO-PHE-(S)ATM-FCO-AEM

To SMO-PHE-(S)ATM(TROC)-FCS-AEM (1.95 g, 1.94 mmol) in anhydrous CH₂ Cl₂(40 mL) at 0° C. was added dichloroacetic acid (0.23 mL, 2.91 mmol)followed by DCC (4.02 g, 19.4 mmol) in CH₂ cl₂ (5 mL) and finallyanhydrous DMSO (3.2 mL, 19.4 mmol). After 2 hours at 0° C., the reactionwas allowed to warm to room temperature and stirred for a further 16hours. After recooling to 0° C. a solution of oxalic acid (3.05 g ) inmethanol (30 mL ) was added dropwise to the reaction mixture. Afterstirring for 20 minutes, the suspension was filtered and evaporatedunder reduced pressure. The residue was taken up in ethyl acetate andrefiltered. After evaporation, this procedure was repeated once more.Evaporation under reduced pressure gave SMO-PHE-(S)ATM(TROC)-FCO-AEM asa viscous yellow oil.

This was dissolved in a mixture of methanol and THF (1:4, 30 mL total).Activated zinc dust (325 mesh) (0.27 g, 3.86 mmol) and excess solidammonium chloride (2 g) was added and the suspension stirred rapidly for30 hours. Further zinc dust (0.27 g) was added after 15 hours. Diethylether (30 mL) was added and the reaction mixture filtered and evaporatedunder reduced pressure to give a white foam. The foam was dissolved in a4:1 mixture of H₂ O and H₃ PO₄ (200 mL) and washed twice with portionsof ethyl acetate (50 mL). The combined organic solution was extractedonce with 20% aqueous H₃ PO₄ (100 mL). The combined aqueous extractswere washed once more with ethyl acetate (50 mL) and then cooled to 0°C. and brought to pH 4.5 by careful addition of ammonium hydroxide(followed with pH meter). The solution was then extracted several timeswith ethyl acetate and dried (Na₂ SO₄). After evaporation under reducedpressure the crude product was obtained as a yellow foam (1.20 g).Column chromatography eluting with 5 to >10% gradient of methanol inethyl acetate on silica gel gave purified product. Combination of therelevant fractions gave a major diastereoisomer (0.50 g) [indicated byHPLC >95% purity as a 98:2 mix as isomers]. The structure was confirmedby NMR and mass spectroscopy. The compound was converted to themethanesulphonic acid salt:

Analyzed for C₃₆ H₅₂ N₈ O₈ F₂ S₂ CH₃ SO₃ H·3.2 H₂ O

Calc'd: C, 45.31; H, 6.41; N, 11.42; S, 9.81

Found: C, 45.31; H, 6.46; N, 11.18; S, 9.49

MH⁺ ; 827.0.

EXAMPLE 24 SMO-PHE-ATG(Z)-CAD

A solution of 1.41 g (4.4 mmol) of SMO-PHE, 0.53 g (4.4 mmol) of Et₃ N,0.62 g (4.4 mmol) of HOBT, and 2.5 g (4.4 mmol) of ATG(Z)-CAD·HCl in amixture of 40 mL of CH₂ Cl₂ and 5 mL of DMF was cooled in ice andtreated with 0.97 g (4.4 mmol) of DCC in 5 mL of CH₂ Cl₂. After 0.5hours at 0° C., the mixture was allowed to stir at room temperature for48 hours. The urea was filtered off and the residue was washed with CH₂Cl₂. The filtrate and the washings were combined, and washedsuccessively with water, saturated NaHCO₃, and brine. Drying and removalof the solvent under reduced pressure gave the crude product. This waspurified via chromatography (SiO₂, CH₂ Cl₂ /CH₃ OH 5%) to give 3.4 g ofproduct. The structure was confirmed by NMR and mass spectroscopy. (M+1ion peak at 829).

EXAMPLE 25 SMO-PHE-ATG-CAD

A solution of 2 g of SMO-PHE-ATG(Z)-CAD in MeOH (75 mL ) containing 0.87g of p-toluene sulfonic acid and 0.5 g of 20To Pd/C was saturated withhydrogen. Methanol was stripped and the residue was taken up in EtOAc.The solution was washed with NaHCO₃, followed by brine, dried andstripped to yield a foam (1.6 g). It was chromatographed (SiO₂ 70 g; CH₂Cl₂ /CH₃ OH 5To) to obtain 1.1 g of the product. Mass spectrum indicate(M+1) ion peak at 695. NMR was consistent with the desired product.

Analyzed for C₃₂ H₅₀ N₆ O₇ S₂ ·0.5 H₂ O

Calc'd: C, 54.60; H, 7.25; N, 11.94

Found: C, 54.60; H, 7.36; N, 12.17

EXAMPLE 26 SMO-PHE-(S)ATE(Z)-CAD

BOC-(S)ATE(Z)-CAD (2.38 g) was added to HCl saturated CH₂ Cl₂ at 0° C.and stirred for 1 hour. The reaction mixture was concentrated to 2.3 gpale yellow foam which was used without further purification. The aminehydrochloride was dissolved in CH₂ Cl₂ at 0° C. and to this added 1.13 gSMO-PHE, 0.74 g DCC, 0.49 g HOBT (1 equivalent each) and 1.00 g DMAP(2.27 equivalents). The reaction was allowed to come to ambienttemperature overnight, then filtered and washed sequentially with 5%aqueous citric acid, saturated NaHCOs, aqueous saturated NaCl and dried(MgSO₄). After concentration, the residue was chromatographed (SiO₂, 95%CHCl₃ 5% MeOH) to give 2.27 g of product. Mass spectrum m/e=857

EXAMPLE 27 SMO-PHE-(S)ATE-CAD

SMO-PHE-(S)ATE(Z)-CAD (2.05 g) was dissolved in methanol to which wasadded 0.40 g 20% Pd/C and 1.18 g tosyl acid. This was put under ahydrogen atmosphere with stirring for 15 hours, then filtered andconcentrated to give 1.21 g off-white foam. This was chromatographed(SiO₂, EtOAc), and 0.51 g product isolated. Mass spectrum m/e=723.3

EXAMPLE 28 SMO-PHE-(S)ATM(Z)-FCS-AEM

The product was synthesized as in Example 1 from 2.8 gSMO-PHE-(S)ATM(Z), 0.554 g HOBT, 0.846 g DCC, and 1.5 g FCS-AEM.Chromatography (silica gel) with O to >10% MeOH gradient in CH₂ Cl₂ gave2.0 g product.

mp=96°-109° C.

Analyzed for C₄₄ H₆₀ F₂ N₈ O₁₀ S₂ ·0.9CH₃ OH

Calc'd: C, 54.36; H, 6.46; N, 11.30

Found: C, 54.06; H, 6.38; N, 11.33

EXAMPLE 29 SMO-PHE-(S)ATM-FCS-AEM

The compound was synthesized as in Example 4 from 1.6 gSMO-PHE-(S)ATM(Z)-FCS-AEM and 0.4 g 20% Pd/C. Silica gel chromatographywith a 5 to 10% methanol gradient in CHCl₃ gave 0.561 g product.mp=103°-115° C.

Analyzed for C₃₆ H₅₄ F₂ N₈ O₈ S₂ ·0.6 CHCl₃

Calc'd: C, 48.81; H, 6.11; N, 12.44

Found: C, 48.74; H, 6.25; N, 12.23

EXAMPLE 30 SMO-PHE-(S)ATM(Z)-CST-BHEAEA

To BOC-ATM(Z)-CST-BHEAEA (3.2 g) in 60 mL CH₂ Cl₂ and 10 mL of methanolwas added HCL (g) for 10 minutes. The solution was evaporated in vacuoto dryness. The residue was dissolved in DMF and treated withdiisopropyl ethyl amine until basic to wet litmus. The new solution wastreated with SMO-PHE (1.34 g), HOBT (0.61 g), and DCC (0.94 g). Themixture was filtered and evaporated to give 4.61 g crude material. Theproduct was purified by silica gel chromatography. Eluting with 19:1,CH₂ Cl₂ :MeOH, gave 1.18 g pure product MS (FAB) M⁺ =945.4. Thestructure was confirmed by IR, NMR, and mass spectroscopy.

EXAMPLE 31 SMO-PHE-(S)ATM-CST-BHEAEA

The product was prepared as in Example 4 from SMO-PHE-ATM(Z)-CST-BHEAEA(1.06 g ), p-TsOH (0.6 g ), 20% Pd/C (0.6 g) in MeOH (40 mL). This gave0.34 g product after silica gel chromatography (19: 1, CH₂ Cl₂ :MeOH).mp=93°-96° C.

Analyzed for C₃₆ H₅₈ N₈ O₉ S₂ ·0.85 CHCl₃

Calc'd: C, 48.50; H, 6.50; N, 12.28

Found: C, 48.56; H, 6.65; N, 12.07

The structure was verified by IR, NMR, and mass spectroscopy.

EXAMPLE 32 SMO-PHE-(S)ATM(TROC)-FCS-OET

The compound was synthesized as in Example 1 from SMO-PHE (0.993 g),HOBT (0.43 g), DCC (0.65 g) and (S)ATM(TROC)-FCS-OET (1.97 g). Thisgave, after silica gel chromatography (6:4, EtOAc:hexane), 1.53 g ofproduct as a white foam. MS, M⁺ =921.

Analysis:

Calc'd: C, 45.68; H, 5.15; N, 9.13

Found: C, 45.92; H, 5.26; N, 8.99

NMR and IR spectra were consistent with assigned structures.

EXAMPLE 33 SMO-PHE-(S)ATM(TROC)-FCO-OET

The compound was synthesized as in the first half of Example 23 fromSMO-PHE-(S)ATM(TROC)-FCS-OET (1.56 g), DMSO (3.5 mL), dichloroaceticacid (0.073 mL), and DCC (3.65 g). This gave after silica gelchromatography (EtOAc:hexane) 1.4 g of product as a white foam. MS, M⁺=919. NMR is consistent with proposed structure.

EXAMPLE 34 SMO-PHE-(S)ATM-FCO-OET

The compound was prepared as in the second half of Example 23 fromSMO-PHE-(S)ATM(TROC)-FCO-OET (1.32 g), ammonium chloride (0.7 g) and Zndust (0.4 g). This gave, after silica gel chromatography (EtOAC:hexane(1:1)), 0.7 g of product as a white foam. NMR, IR, and mass spectra allare consistent with the structure.

Analysis:

Calc'd: C, 51.68; H, 5.98; N, 11.30

Found: C, 51.39; H, 6.05; N, 10.89

EXAMPLE 35 SMO-PHE-ATG(Z)-FCS-AEM, Isomer A

The compound was synthesized as in Example 1 from SMO-PHE (0.74 g), HOBT(0.32 g), DCC (0.5 g), and ATG(Z)-FCS-AEM, Isomer A (1.5 g). This gave,after silica gel chromatography (3-10% MeOH in 1:1 (EtOAc:CH₂ Cl₂)),1.26 g of product as a foam.

Analyzed for 0.2CH₂ Cl₂

Calc'd: C, 53.72; H, 6.05; N, 11.49

Found: C, 53.38; H, 6.03; N, 11.49

EXAMPLE 36 SMO-PHE-ATG-FCS-AEM, Isomer A

The compound was synthesized as in Example 4 fromSMO-PHE-ATG(Z)-FCS-AEM, Isomer A (1.0 g), 20% Pd/C (0.25 g), andp-toluenesulfonic acid hydrate (0.6 g). This gave, after silica gelchromatography (5% MeOH in 1:1 (EtOAc:CH₂ Cl₂)), 0.1 g of product.

Analyzed for ·H₂ O

Calc'd: C, 50.51; H, 6.50; N, 13.45

Found: C, 50.46; H, 6.55; N, 12.82

NMR and IR spectra were consistent with structure.

MS, M⁺ =815.

EXAMPLE 37 SMO-PHE-ATG(Z )-FCS-AEM, Isomer B

The compound was synthesized as in Example 1 from SMO-PHE (0.84 g), HOBT(0.36 g), DCC (0.57 g), and ATG(Z)-FCS-AEM, Isomer B (1.7 g). This gave,after silica gel chromatography (3-10% MeOH in 1:1 EtOAc:CH₂ Cl₂ ), 1.8g of product as a foam.

Analyzed for 0.2CH₂ Cl₂

Calc'd: C, 53.72; H, 6.05; N, 11.60

Found: C, 53.41; H, 6.19; N, 11.32

NMR and IR were consistent with structure.

EXAMPLE 38 SMO-PHE-ATG-FCS-AEM, Isomer B

The compound was synthesized as in Example 4 fromSMO-PHE-ATG(Z)-FCS-AEM, Isomer B (2.5 g), 20% Pd/C (0.5 g) andp-toluenesulfonic acid (1.5 g). This gave, after silica gelchromatography, 1.0 g of the product as a foam.

Analysis:

Calc'd: C, 51.62; H, 6.38; N, 13.76

Found: C, 51.39; H, 6.56; N, 12.85

NMR and IR spectra were consistent with the structure. MS, M⁺ =815.

INTERMEDIATES FOR EXAMPLES 1-38 DMSA-PHE

A solution of PHE (3.3 g) in 1 N NaOH (20 mL) was treated with asolution of N,N-dimethylsulfamyl chloride (2.3 mL) in THF (20 mL) andstirred vigorously at 25° C. for 3 hours. The reaction mixture was thentreated with additional 1 N NaOH (20 mL) and N,N-dimethylsulfamylchloride (2.3 mL) and stirred 3 hours further at 25° . Finally 1 N NaOH(20 mL) and diethyl ether (80 mL) were added. The mixture was shaken andthe aqueous layer was separated and acidified to pH 1 by addition of 1 NHCl (25 mL). The product was extracted into ethyl acetate, the solutiondried over MgSO₄, and evaporated to give a gum which slowly solidified(4.0 g). The structure was confirmed by NMR spectroscopy.

B SMO-PHE

Prepared as above, substituting morpholino-sulfamyl chloride (preparedaccording to the method of R. Wegler and K. Bodenbenner, Annallen derChemie, 624, 25 (1959)) for N,N-dimethylsulfamyl chloride. The productis a solid, mp 151°-153° C.

C DMSA-TYR(OME)

Prepared as above, substituting TYR(OMe) for PHE. The product wasisolated as its dicyclohexylamine salt, mp 157°-159° C.

D DMS A-TYR (OME)

Prepared as above, substituting TYR(OME) for PHE. The structure wasconfirmed by NMR spectroscopy.

E BOC-ATG(Z)

Prepared as in reference J. Chem. Soc. Perkin Trans. I, p 1227 (1984).

To a solution of ethyl 2-aminothiazol-4-ylacetate (27.4 g) in water (200mL) was added K₂ CO₃ until pH=9) (wet litmus). To this was added BOC₂ O(26.0 g) in THF (150 mL) and the pH continually adjusted to 9 with K₂CO₃. The mix stirred for 24 hours. The basic solution was then extractedwith ethyl acetate (2×400 mL). The organics were washed with brine (3090mL) and dried over MgSO₄. Evaporation gave a brown oil which wasrecrystallized from ethyl acetate (100 mL) and petroleum ether (600 mL).This gave 23.5 g of a beige solid(ethyl-N-BOC-2-aminothiazol-4ylacetate). This ester (15.07 g) was thendissolved in a mixture of THF (65 mL), CH₂ Cl₂ (100 mL) and saturatedNaHCO₃ solution (700 mL). To this was added benzylchloroformate (21.3 g)and the mixture vigorously stirred for 18 hours. The solution wasdiluted with water (100 mL) and ethyl acetate (150 mL). The organicswere separated, washed with water (2×400 mL), and evaporated at reducedpressure to give an oil which was dissolved in EtOH (150 mL) and treatedwith a solution of potassium hydroxide (14 g) in water (100 mL). Themixture was stirred for 1.5 hours and diluted with water (350 mL). Thesolution was washed with ether (2×250 mL) and the ethereal solutiondiscarded. The aqueous solution was made acidic (pH=3, wet litmus) withcitric acid and cooled to precipitate. Collection of the solid gave 16.8g of product.

F BOC-ATM(Z)

Prepared in a similar manner as BOC-ATG(Z) from ATM·2HCl which wasprepared as in reference Chem. Ber., Vol. 97, p 1767 (1964).

Sodium metal (0.64 g) was dissolved in ethanol (75 mL) and the solutiontreated with diethyl acetamido malonate (6.08 g) and stirred for 0.5hours. This was then treated with2-acetamidothiazol-4-yl-1-chloromethane (5.3 g) and sodium iodide (4.17g). The mixture was stirred under nitrogen for 24 hours. The solutionwas filtered free of solids. The solids were triturated with water (150mL). The mixture was again filtered to collect solid and washed withwater (2×150 mL). This solid (2.75 g) was dissolved in concentratedhydrochloric acid (40 mL) and warmed to reflux for 3 hours.

The solution was evaporated in vacuo and coevaporated with ethanol (2×50mL). The resultant solid was triturated with ether (100 mL) and filteredto give 1.91 g of product as a white solid.

G BOC-ATM (Z)-CAD

The compound was prepared as in Example 1 from BOC-ATM(Z) (6.3 g), HOBT(2.02 g), DCC (3.1 g), CAD·HCl (4.19 g) and Et₃ N: (1.5 g). This gave5.7 g of the product as a mixture of diastereomers as a white foam, mp95°-100° C. The structure was confirmed by NMR and mass spectroscopy.

H ATM(Z)-CAD

The compound was prepared by saturating a solution of BOC-ATM(Z)-CAD(5.35 g) in CH₂ Cl₂ (70 mL) and MeOH (10 mL) with HCl gas. The solutionwas stirred at room temperature for 2 hours and then evaporated in vacuoto dryness. The residue was dissolved in EtOAc (75 mL) and washedsuccessively with saturated NaHCOs (2×100 mL) and saturated saltsolution (100 mL). The organic phase was dried over MgSO₄ and evaporatedin vacuo to give 3.8 g of product as a mixture of diastereomers as alight yellow foam, mp 84°-91° C. The structure was confirmed by NMR andmass spectroscopy.

I BOC-ATM (Z)-CST-AEM

The compound was synthesized as in Example 1 from BOC-ATM(Z) (4.21 g),HOBT (1.35 g), DCC (2.06 g) and CST-AEM (3.27 g). This gave 4.6 g ofproduct as a mixture of diastereomers as a white foam, mp 97°-100° C.The structure was confirmed by NMR and mass spectroscopy.

J ATM(Z)-CST-AEM

The compound was prepared as in ATM(Z)-CAD from BOC-ATM(Z)-CST-AEM (4.4g). This gave 3.78 g of product as a mixture of diastereomers as a whitefoam, mp 80°-83° C. The structure was confirmed by NMR and massspectroscopy.

K BOC-ATM (Z)-STA-MBA

The compound was synthesized as in Example 1 from BOC-ATM(Z) (1.0 g),HOBT (0,338 g), DCC (0.516 g), and STA-MBA (0.733 g). This gave 1.25 gof product as a mixture of diastereomers as a white foam. The structurewas confirmed by NMR and mass spectroscopy.

L ATM (Z)-STA-MBA

The compound was synthesized as in ATM(Z)-CAD above fromBOC-ATM(Z)-STA-MBA (1.25 g). This gave 0.9 g of product as a mixture ofdiastereomers as a light yellow foam. The structure was confirmed by NMRand mass spectroscopy.

M BOC-ATG (Z)-STA-MBA

The compound was synthesized as in Example 1 from BOC-ATG(Z) (6.1 g),HOBT (2.16 g), DCC (3.3 g), and STA-MBA (3.7 g) . This gave 8.8 g ofproduct as a mixture of diastereomers as a white foam, mp 86°-91° C. Thestructure was confirmed by NMR and mass spectroscopy.

N ATG(Z)-STA-MBA

The compound was prepared as in ATM(Z)-CAD from BOC-ATG(Z)-STA-MBA (3.17g). This gave 2.2 g of product as a mixture of diastereomers as a lightyellow foam, mp 76°-83° C. The structure was confirmed by NMR and massspectroscopy.

BOC-(S)ATM-OBZL·HCL

N-BOC-Aspartatic acid, α-benzyl ester (40 g, 0.124 mol) in EtOAc (1 L)was treated at 0° C. with N-methylmorpholine (13.8 g, 0.136 mol) andisobutyl chloroformate (18.6 g, 0.136 mol). The mixture was stirred at0°-10° C. for 3 hours. The mixture was filtered free of precipitate andtreated with a solution of diazomethane [(˜0.175 mol) freshly distilledfrom diazald (53 g)] in ether (˜500 mL). The mixture was stirred for 16hours under a N₂ stream. The solution was washed with saturated saltsolution (500 mL) and evaporated in vacuo to give the diazoketone as adark oil. This oil was dissolved in ether (400 mL) and carefully treatedwith HCl gas. The gas treatment stopped when the pH of the solutionreached 2 (wet litmus), approximately two to 8 minutes. The solution wasthen immediately treated with a solution of saturated sodium bicarbonate(600 mL). The organics were washed with saturated salt solution (200 mL)and dried over MgSO₄. The organics were evaporated in vacuo to give 44.4g of the chloroketone as a tan solid. This was dissolved in acetone (225mL) and treated in portions with thiourea (7.6 g, 0.1 mol). The solutionwas stirred at room temperature for 24 hours. The mixture was filteredto collect solid, the solid washed with acetone (2×75 mL) and dried invacuo to give 20.6 g 10 of product as a white solid, mp=144°-146° C. Thestructure was confirmed by NMR and mass spectroscopy.

P BOC-(S)ATM(Z)

Prepared in a manner similar to BOC-ATM(Z) from BOC-(S)ATM-OBZL·HC1 asin ref. Chem. Ber., Vol. 97, p. 1767 (1964).

BOC-(S)ATM(Z)-CAD

To BOC-(S)ATM(Z)-OBZL·HC1 (2.06 g) in methanol (35 mL) was added asolution of NaOH (0.6 g) in water (10 mL). The solution was stirred atroom temperature for 4 hours and then taken to pH=6 (wet litmus) with 1NHCl. The solution was evaporated in vacuo and dissolved in DMF (20 mL).This solution was treated at 0° C. sequentially with Et₃ N (1.51 g),HOBT (0.667 g), DCC (1.03 g), and CAD (1.22 g). The mixture was stirredfor 72 hours. The mixture was filtered free of solids and the solventevaporated in vacuo. The residue from evaporation was dissolved in EtOAc(100 mL) and washed sequentially with saturated sodium bicarbonate (100mL) and saturated salt solution. The organics were dried over MgSO₄ andevaporated in vacuo to give a yellow foam. The foam was chromatographedover silica gel to give the product as a white solid, 1.2 g. Thestructure was confirmed by NMR and mass spectroscopy.

R (S)ATM(Z)-CAD·2HCl

To BOC-(S)ATM(Z)-CAD (1.1 g) in a mix of CH₂ Cl₂ (75 mL) and MeOH (15mL) was added HCl (gas) and the solution stirred at room temperature for3 hours. The solution was evaporated in vacuo to give the product, whichwas used without further purification. The structure was confirmed byNMR and mass spectroscopy.

S BOC-(S)ATE-OBZL

The compound was synthesized as in intermediate O BOC-(S)-ATM-OBZL from42.2 g of N-BOC-GLU-OBZL 18.6 g i-butyl chloroformate, 13.8 g N-methylmorpholine, and excess Diazomethane. Silica gel chromatography gave 15.2g of product. The structure was confirmed by NMR, IR and massspectroscopy.

T BOC-(S)ATE(Z)-OBZL

The compound was synthesized as in intermediate BOC-(S)ATM(Z)-OBZL from14.17 g BOC-(S)ATE-OBZL, and 15.4 g Z-C1. Extraction workup with EtOAcgave after evaporation 18.9 g of product. The structure was confirmed byNMR spectroscopy.

U BOC-(S)ATE(Z)

A mixture of 18.9 g BOC-(S)-ATE(Z)-OBZL and 7 g NaOH were stirred in amixture of 100 mL methanol and 20 mL water for 6 hours. The mixture wasevaporated and treated with 100 mL 1N NaOH, then washed with 100 mLdiethyl ether. The aqueous solution made acidic with 6N HCl andextracted with ethyl acetate (2×150 mL). Drying over MgSO₄ andevaporation gave 9.3 g of product. The structure was confirmed byspectroscopy.

V BOC-(S)ATE(Z)-CAD

The compound was synthesized as in Example 1 from 9.3 g BOC-(S)ATE(Z),2.7 g HOBT, 4.12 g DCC and 4.9 g CAD. Silica gel chromatography withethyl acetate gave 7.6 g of product. The structure was confirmed by NMRand mass spectroscopy.

W TROC-PIP·HCl

A solution of 2.37 mL Cl₃ CCH₂ OCOCl (17.22 mmol) in 65 mL CH₂ Cl₂ wasadded dropwise to a chilled solution of 15.00 g piperazine (174.13 mmol,a 10-fold excess) in 75 mL MeOH under N₂ atmosphere and allowed to stirat room temperature for 3 hours. The reaction mixture was diluted withMeOH and evaporated to a wet solid. The residue was taken up in H₂ O andextracted with EtOAc. The organic layer was dried over MgSO₄ andevaporated. The resulting oil was dissolved in 40 mL Et₂ O and treateddropwise with a 1.0 M HCl solution in 40 mL Et₂ O under N₂ atmosphere togive 2.45 g of the product as a white solid. mp 209°-213° C.

X TROC-SPI-Cl

A solution of 9.75 g TROC-PIP hydrochloride (32.72 mmol) in 20 mL H₂ Owas mixed with 40 mL CH₂ Cl₂ and was cooled in ice, treated dropwise bya solution of 48.60 g 5.25% NaOC1 (34.03 mmol) in H₂ O and stirred onice for 50 minutes. The organic layer was separated and dried overMgSO₄. The resulting solution was added to a -78° solution of 30.00 gSO₂ in 20 mL CH₂ Cl₂, and catalytic amount of Cl₂ (6 drops) wascondensed into the reaction mixture under N₂ atmosphere. The reactionmixture was allowed to warm up to room temperature over 24 hours. Thesolvent was evaporated, and the residue was partitioned between CH₂ Cl₂and 0.25 M K₂ PO₄, pH=7 buffer and washed with 10% Na₂ S₂ O₃. Theorganic layer was dried over MgSO₄ and evaporated to give 8.02 g of theproduct as a golden brown solid. The structure was confirmed by NMRspectroscopy.

Y BOC-ATG(Z)-CAD

A solution of 2.2 g (8 mmol) of CAD-hydrochloride, 1 g (10 mmol) of Et₃N, 1.1 g (8 mmol) of HOBT, and 3.3 g (8 mmol) BOC-ATG(Z) in 45 mL of DMFwas cooled in ice and treated with 1.7 g (8 mmol) of DCC in 10 mL ofDMF. After 0.5 hour at 0° C., the mixture was allowed to stir at roomtemperature for 48 hours. DMF was distilled and the residue was treatedwith EtOAc. The urea was filtered off and the residue was washed withadditional quantities of EtOAc. The filtrate and the washings werecombined, and washed successively with water, saturated NaHCO₃, andbrine. Drying and removal of the solvent under reduced pressure gave thecrude product. This was purified via chromatography (SiO₂, CH₂ Cl₂ /CH₃OH 10%) to give 6 g of product. The structure was confirmed by NMRspectroscopy.

Z ATG(Z )-CAD·HCl

A solution of 6 g of BOC-ATG(Z)-CAD in 60 mL of CH₂ Cl₂ was saturatedwith HCl (g), and allowed to stand at room temperature for 2 hours. Thesolution was evaporated to give a foam. The structure was confirmed by ¹H NMR and mass spectrum (M+1 peak at 533). This material was used as isfor coupling.

AA BOC-(S)ATM(TROC)-FCS-AEM

To BOC-(S)ATM(TROC) (2.60 g, 5.63 mmol) and HOBt (0.76 g, 5.63 mmol) inanhydrous DMF (30 mL) at 0° C. was added DCC (1.16 g, 5.63 mmol) in DMF(10 mL) followed by FCS-AEM (2.04 g, 5.63 mmol) in DMF (10 mL) and thereaction stirred at 0° C for 2 hours before warming to room temperature.After 16 hours the reaction was filtered and the solvent evaporatedunder reduced pressure. The residue was taken up in ethyl acetate (50mL) and washed with saturated aqueous sodium bicarbonate solution,water, and brine, respectively. After drying (Na₂ SO₄), the solvent wasevaporated and the crude material chromatographed on silica gel elutingwith a gradient of 3 to >5% methanol in CH₂ Cl₂. The product wasobtained as a white foam (3.0 g, 66%) and the structure confirmed by NMRand mass spectroscopy; MS(FAB):MH⁺ 807.2.

BB (S)ATM(TROC)-FCS-AEM

BOC-(S)ATM(TROC)-FCS-AEM (2.20 g, 0.27 mmol), in a mixture of CH₂ Cl₂(30 mL) and methanol (10 mL) was subjected to a stream of HCl gas atroom temperature for 15 minutes. After stirring for a further 20minutes, the solvent was evaporated and the residue taken up inchloroform and reevaporated. This procedure was repeated twice morebefore addition of ethyl acetate to the crude product. The solution waswashed with saturated aqueous bicarbonate solution to generate the freebase. The aqueous layer was separated and extracted twice with ethylacetate. The combined organic extracts were washed with brine and dried(Na₂ SO₄). After evaporation of the solvent the free amine was obtainedas a white foam (1.90 g, 99%). The structure was confirmed by NMR andmass spectroscopy; MS(FAB) M⁺ 707.0.

CC BOC-FCS-AEM

To BOC-FCS (4.10 g, 12.0 mmol ) and HOBt (1.58 g, 12.0 mmol) in DMF (100mL) at 0° C. was added DCC (2.41 g, 12.0 mmol) in DMF (20 mL). After 5minutes, AEM (1.53 mL) in DMF (10 μL) was added and the reaction stirredfor 2 hours before warming to room temperature and stirring for afurther 16 hours. The mixture was filtered and the solvent evaporatedunder high vacuum. The residue was taken up in ethyl acetate and washedwith saturated sodium bicarbonate solution, water, and brinerespectively. After drying (Na₂ SO₄) the solvent was evaporated and thecrude product purified by column chromatography on silica gel elutingwith 5% to 7% methanol in dichloromethane. The product was obtained as awhite foam (3.38 g, 63%) and the structure confirmed by NMR and massspectroscopy: MS(FAB)MH⁺ (464 ).

DD BOC-(S)ATM(TROC)-OBZL

A solution of 9.56 g (25 mmol) BOC-(S)ATM-OBZL was suspended in CH₂ Cl₂and cooled in an acetone/ice bath. Added to this was 7.74 g DMAP (2.5equivalent); goes into solution. TROC-Cl of 8.40 mL (12.34 g, 2.3equivalent) was added dropwise. After 4 hours the solution is washedsequentially with water, 0.1N HCl, saturated aqueous NaCl, and dried(MgSO₄), then concentrated. The residue is chromatographed (400 g SiO₂,eluting 1:1 hexane/ethyl acetate. The product was isolated as acolorless foam, 12.80 g. Mass spectrum m/e=552.

EE BOC-(S)ATM(TROC)

BOC-(S)ATM(TROC)-OBZL of 3.60 g (17.6 mmol) was dissolved in 100 mLmethanol at room temperature. KOH (1.16 g) (85%, 2.7 equivalent) wasdissolved in 10 mL water and added to the solution. Stir for 3.5 hours.An additional 50 mL of water was added, the reaction partiallyconcentrated, then extracted with EtOAc. These organics were discarded.The aqueous solution was then rendered acidic (6N HCl formsprecipitate), and extracted again with EtOAc. These organics were washedwith saturated aqueous NaCl dried (MgSO₄) and concentrated to give 2.54g product as a waxy white solid. Mass spectrum m/e=462

FF BOC-(S)ATM(TROC)-CAD

BOC-(S)ATM(TROC) 27.25 g (58.9 mmol) was dissolved in EtOAc and cooledin ice bath. CAD (14.33 g, 58.9 mmol) was added, resulting in asuspension. To this was added 7.98 g HOBT and 12.13 g DCC (oneequivalent each), and the reaction mixture allowed to come to ambienttemperature overnight. The reaction was filtered, and the filtratewashed sequentially with 5% aqueous citric acid, saturated NaHCOs,saturated NACl then dried (MgSO₄) and concentrated to afford 31.0 gproduct. This was recrystallized in 150 mL boiling EtOAc. The first cropyielded 20.50 g, and a second crop 2.43 g product. An additional 6.59 gproduct was recovered by washing the original filter cake with acetoneand concentrating this filtrate. Mass spectrum m/e=689

GG BOC-(S)ATM(TROC)-CDH

BOC-(S)ATM(TROC) (2.50 g, 5.4 mmol) was dissolved in methylene chlorideand the solution cooled in an ice bath. CDH (1.25 g, 0.95 equivalent)was added, followed by 0.70 g HOBT, 1.06 g DCC, and 1.26 g DMAP. Themixture was allowed to come to ambient temperature overnight, thenfiltered and the filtrate washed sequentially with 1N HCl, saturatedNaHCO₃ a, saturated NaCl, and dried (MgSO₄). After concentration, theresidue was chromatographed (SiO₂, EtOAc) to give 0.76 g product. Thiswas used without further purification in the next reaction.

HH SMO-PHE-(S )ATM(Z)

SMO-PHE-(S)ATM(Z)-OBZL (5.6 g) and 0.64 g NaOH were stirred in a mix of70 mL methanol and 15 mL water for 5 hours. The mixture was evaporatedin vacuo and the residue partitioned between 100 mL EtOAc and 100 mL 2NHC1. The organics were separated and dried over MgSO₄. Evaporation ofsolvents gave 4.5 g of product.

II (S)ATM(Z)-OBZL-HCl

BOC-(S)ATM(Z)-OBZL (10.8 g) was dissolved in 8 mL MeOH and 50 mL CH₂ Cl₂and treated with HCl(g) at room temperature for 3 hours. Evaporationgives 8.8 g of product as the HCl salt. The structure was confirmed byNMR, IR, and mass spectroscopy.

JJ SMO-PHE-(S)ATM(Z)-OBZL

The compound was synthesized as in Example 1 from 3.14 g SMO-PHE, 1.35 gHOBT, 2.06 g DCC, 2.02 g Et₃ N and 4.3 g (S)ATM(Z)-OBZL. Chromatographyon the Waters prep 500A using 5% methanol/CH₂ Cl₂ gave the product as ayellow solid, used as is.

KK TROC-SPI-PHE

To a suspension of 5.09 g PHE (30.81 mmol) in 20 mL MeOH was added 12.97mL of 5% Me₄ NOH/MeOH (30.81 mmol) and the mixture stirred until asolution was obtained. The mixture was diluted with toluene andevaporated to a foam and dried at 0.05 mm at room temperature for 2hours. To a suspension of the foam in 30 mL dry THF was added 60 mL dryi-PrOH and 5.49 g TROC-SPI-Cl. The mixture was allowed to stir at roomtemperature under N₂ atmosphere for 16 hours. The solvent wasevaporated, and the residue was partitioned between CH₂ Cl₂ and 1N HCland washed with 1N HCl. The product was extracted from the organic layerinto 0.3 N NaOH which was immediately acidified to pH=1 withconcentrated HCl and reextracted with EtOAc. The EtOAc layers werewashed with 1N HCl saturated NaCl dried over MgSO₄ and evaporated toobtain 3.59 g of the product as a golden brown solid. The structure wasconfirmed by NMR spectroscopy.

LL FCS-AEM

To BOC-FCS-AEM (5.17 g) in dry CH₂ Cl₂ (100 mL) was added 6 mL of TFA atRT and the reaction stirred for 6 hours. After concentration the residuewas treated with saturated NaHCO₃ solution and the product extractedinto ethyl acetate and washed with brine and dried (Na₂ SO₄). Driedunder high vacuum for several hours to give the product as a white foam,3.65 g. Compound confirmed by NMR spectroscopy.

MM BOC-(S)ATM(Z)-CYSTA-BHEAEA

The product was prepared as in Example 1 from BOC-(S)ATM(Z) (5 g), HOBT(1.68 g), DCC (2.6 g) and CST-BHEAEA (4.1 g) in DMF (25 mL). This gave3.26 g of product after silica gel chromatography (19: 1, CH₂ Cl₂:MeOH).

Analyzed for C₃₆ H₅₆ N₆ O₉ S·0.8 CHCl₃

Calc'd: C, 52.34; H, 6.78; N, 9.95

Found: C, 52.52; H, 6.91; N, 9.91

NN BOC-FCS-OET

To a suspension of Zn dust (15.3 g) in THF (300 mL) was added onecrystal of I₂ and the mixture brought to reflux. To this was addedsequentially bromodifluoro ethyl acetate (0.2 mL) followed by a mixtureof the same ester (25.3 g) and (S)-BOC-cyclohexyl alanol (20 g) in THF(100 mL). The mixture was filtered free of insoluble material and thesolvents evaporated at reduced pressure. The residue was dissolved inethyl acetate and the pH adjusted with 1M KHSO₄ solution until pH=2.This solution was filtered free of solids and the organics separatedfrom the filtrate. The aqueous solution was extracted with ethyl acetate(2X) and the combined organic layers washed with brine, dried overmagnesium sulfate, and evaporated in vacuo to give a yellow solid. Thesolid was triturated with 20% ethyl acetate in hexane to give theproduct as a white solid, 6.5 g. NMR and IR were consistent with thestructure.

OO FCS-OET·HCl

To BOC-FCS-OET (5.3 g) in methanol (250 mL) was added HCl gas, gasdispersion tube, until the solution was saturated. The solution wasstirred at room temperature for 2 hours and evaporated at reducedpressure to give the product as a white solid, 4.5 g. NMR was consistentwith structure.

PP BOC-(S)ATM(TROC)-FCS-OET

The compound was synthesized as in Example 1 from BOC-(S)ATM(TROC) (5.90g), HOBT (1.73 g), DCC (2.64 g), Et₃ N (1.43 mL), and FCS-OET·HCl (4.03g). This gave, after silica gel chromatography (4% MeOH/CH₂ Cl₂) 2.55 gof product. NMR spectra were consistent with the structure.

QQ (S)ATM(TROC)-FCS-OET

To BOC-(S)ATM(TROC)-FCS-OET (2.54 g) in EtOH (25 mL) was added CH₂ Cl₂(200 mL) and the solution saturated with HCl gas. The mixture wasallowed to stand overnight and then evaporated at reduced pressure. Thissolid was partitioned between EtOAc (250 mL) and saturated NaHCO₃solution (200 mL). The organic phase was washed with brine and driedover MgSO₄. Evaporation of solvents gave 1.97 g of product.

Analysis:

Calc'd: C, 40.01; H, 4.88; N, 8.48

Found: C, 38.03; H, 4.94; N, 8.50

RR and SS BOC-ATG(Z)-FCS-AEM (Isomer A) RR BOC-ATG(Z)-FCS-AEM (Isomer B)SS

The compounds were synthesized as in Example 1 from BOC-ATG(Z) (3.3 g),HOBT (1.1 g, DCC (1.7 g), and FCS-AEM (2.9 g). This gave after silicagel chromatography (3-5% MeOH/CH₂ Cl₂), 1.9 g of isomer A (fast eluting)and 1.9 g of isomer B (slow eluting). Both compounds gave NMR, IR, andmass spectra consistent with structures.

TT ATG(Z)-FCS-AEM, Isomer A

The compound was synthesized as in intermediate H, fromBOC-ATG(Z)-FCS-AEM, Isomer A (1.9 g). This gave 1.5 g of free base whichwas used as is.

UU ATG(Z)-FCS-AEM, Isomer B

The compound was synthesized as in intermediate H, fromBOC-ATG(Z)-FCS-AEM, Isomer B (1.9 g). This gave 1.7 g of free base whichwas used as is.

We claim:
 1. A process for the preparation of a compound of the formula##STR30## or a pharmaceutically acceptable acid addition salt thereofwherein A is 2-benzyl-3-(t-butylsulfonyl)propionyl, ##STR31## wherein Rand R₁ are each independently hydrogen or straight or branched chainlower alkyl which is unsubstituted or substituted by one or twohydroxy,one or two amino groups, or ##STR32## wherein ##STR33## is asaturated ring containing four carbon atoms atoms wherein Z is O, or NRwherein R is as above; B is absent, PHE, or TYR(OME) with the provisothat when A is 2-benzyl-3-(t-butylsulfonyl)propionyl, B is absent; C isFCS, FCO, CAd, or CDH; D is absent; E is hydrogen; n is an integer from0 to 2;X and Y are each independently O, S, N or NH and at least one ofX and Y must be N; X and Y cannot both be N which comprises: a) reactingan N-protected amino acid with an amine to produce the correspondingamide, b) deprotecting the N-protecting group of said amide and couplingit with an acid to produce a dipeptidyl like amide, and c) furtherdeprotecting the side chain functions on the amide to produce a compoundof formula I and converting, if desired, to a pharmaceuticallyacceptable salt thereof.
 2. A process for the preparation of a compoundof the formula ##STR34## or a pharmaceutically acceptable acid additionsalt thereof wherein A is 2-benzyl-3-(t-butylsulfonyl)propionyl,##STR35## wherein R and R₁ are each independently hydrogen or straightor branched chain lower alkyl which is unsubstituted or substituted byone or two hydroxy,one or two amino groups, or ##STR36## ##STR37## is asaturated ring containing four carbon atoms atoms wherein Q is O, or NRwherein R is as above; B is absent, PHE, or TYR(OME) with the provisothat when A is 2-benzyl-3-(t-butylsulfonyl)propionyl, B is absent; C isFCS or FCO; D is absent; E is hydrogen; n is an integer from 0 to 2; Xand Y are each independently O, S, N or NH and at least one of X and Ymust be N; X and Y cannot both be N which comprises:a) reacting an aminoacid ester with TROC protection on the side chain with TROC protectionon the side chain with an N-protected amino acid to produce a dipeptideester which is then hydrolyzed to the corresponding dipeptide acid, b)coupling the product of step a) with an amine selected from the groupconsisting of FCS-or FCO to produce the TROC-protected compound offormula I, and c) deprotecting further to produce a compound of formulaI and converting, if desired, to a pharmaceutically acceptable saltthereof.